ShcA tyrosine phosphorylation sites can replace ShcA binding in signalling by middle T-antigen

Abstract

ShcA and Grb2 are crucial components in signalling by most tyrosine kinase‐associated receptors. How ever, it is not clear whether Grb2 bound directly to the receptor is equivalent to Grb2 associated via ShcA. We have used signalling stimulated by the middle T‐antigen (MT) of polyoma virus to address this question. The two known Grb2‐binding sites from murine ShcA, 313Y and 239/240YY, could functionally replace the MT ShcA‐interacting region in transformation assays using Rat2 fibroblasts. This demonstrates that signal output from membrane‐bound ShcA requires only these two sequences and the ShcA‐binding site in MT does not recruit other signalling molecules. Two standard Grb2‐interacting sequences, either from the EGF receptor or the ShcA 313Y region, could not replace the requirement for ShcA binding to MT, indicating an enhanced role for the ShcA 239/240YY motif. Sos1 and the docking protein Gab1 are brought into the MT complex through Grb2 association and this may be more effective using the 239/240YY sequence.

Lessons in Signaling and Tumorigenesis from Polyomavirus Middle T Antigen

“…The binding of Grb2 allows the recruitment of additional proteins such as the Ras exchange factor SOS (207). This is consistent with observations that MT activates Ras (277).…”

“…The association of Shc and Grb2 is driven by MT (28,86), and Grb2 is also found in MT complexes (207). Grb2 function is important for MT tumorigenesis.…”

“…When the sequence around residue 250 of MT is replaced with ShcA binding sites known to bind Grb2 (the Y317 [Y313] ShcA site [246] and the Y239/Y240 site [306]), MT bearing the 239/240 sequence was better able to transform Rat-2 cells than MT bearing the 313 sequence (207). However, these mutants, which allow binding of amounts of Grb2 matching the wild type, were still less active than MT.…”

The tyrosine kinase Abl is required for Src-transforming activity in mouse fibroblasts and human breast cancer cells

“…Nevertheless, these phosphorylations were required for Src-mT-induced foci as shown by the inhibitory effects of Shc and Stat3 mutants in which these residues have been replaced by Phe (Shc2Y/2F and Stat3Y705F) (Figure 2b). Tyr315 is another reported phosphorylation site of Shc required for Src-mTinduced foci (Nicholson et al, 2001). However, Imatinib did not affect Shc tyrosine phosphorylation at all (Figure 2a), suggesting that Abl does not phosphorylate this residue.…”

“…Tyrosine phosphorylation of Stat3 and Shc regulate Srcinduced cell transformation (Bromberg et al, 1998;Nicholson et al, 2001). Whether these phosphorylations require Abl in Src-transformed cells was next addressed.…”